Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains

ABSTRACT

It has unexpectedly been found that a low alcohol or alcohol-free beverage, with a flavor profile very close to a beer of at least 4% (vol/vol) alcohol, can be produced by using Pichia kluyveri yeast strains. In particular, Pichia kluyveri yeast strains only use the glucose in the wort, and have the ability of converting this substrate into a high concentration of specific flavor compounds, which are normally produced by Saccharomyces ssp. yeast strains used for the brewing of beer. In this way the Pichia kluyveri yeast strains can be used to produce either a low alcohol or alcohol-free beverage, depending on the glucose levels in the wort. The main flavor compounds produced by Pichia kluyveri in the fermentation of wort are isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/413,246, filed Jan. 23, 2017 (now U.S. Pat. No. 10,415,007), which isa continuation of U.S. application Ser. No. 14/773,239, filed Sep. 4,2015 (now U.S. Pat. No. 9,580,675), which is the U.S. National Stage ofInternational Application PCT/EP2014/054415, filed Mar. 7, 2014, andclaims priority to European Patent Application No. 13158261.1 filed Mar.7, 2013.

FIELD OF THE INVENTION

The present invention relates to the field of preparing a low-alcohol oralcohol-free beverage by fermentation. Specifically, the inventionrelates to a method of preparing a low-alcohol or alcohol-free beveragewith enhanced levels of desirable fermentation-derived flavor compoundscomprising a step of fermentation of a wort with a Pichia kluyveri yeaststrain to obtain a low-alcohol or alcohol-free beverage, a low-alcoholor alcohol-free beverage obtainable by the method and a low-alcohol oralcohol-free beverage comprising a high concentration of specific flavorcompounds, such as isoamyl acetate and ethyl octanoate.

BACKGROUND OF THE INVENTION

Due to the increasing demand for healthier food and beverages, thereduction of ethanol in alcoholic beverages, especially beer and wine,is of considerable commercial interest.

Low-alcohol, alcohol-free or reduced-alcohol beers (also called lightbeer, no-alcohol, non-alcoholic beer, small beer, small ale, near-beer)are beers with no alcohol or a low alcohol content which aim toreproduce the full flavor of standard beers which normally contain morethan 4% (vol/vol) alcohol. In most of the EU countries beers with lowalcohol content are divided into alcohol-free beers containing no morethan 0.5% (vol/vol) alcohol, and into low-alcohol beers with no morethan 1.2% (vol/vol) alcohol. In the United States alcohol-free beermeans that there is no alcohol present, while the upper limit of 0.5%(vol/vol) alcohol corresponds to so-called non-alcoholic or near-beer(Montanari et al. 2009).

The conversion of wort into an alcoholic beer is the result of afermentation process by Saccharomyces ssp. yeast resulting in theproduction of alcohol and fermentation-derived 35 flavor compounds, suchas esters (e.g. isoamyl acetate) and higher alcohols. Isoamyl acetate isa key flavor compound in beer (Saerens et al. 2010). The combination ofthe flavor of the raw materials, mostly malt and hops, together with thefermentation process, results in the unique flavor and taste of beers(Swiegers et al. 2007, Saerens et al. 2010).

Today beers with low alcohol contents are often made by producingfull-strength beer (with an alcohol concentration of above 4% (vol/vol))and then removing the alcohol by physical processes (boiling off thealcohol/distillation or reverse osmosis). In the case of somenon-alcoholic or alcohol-free beers no fermentation takes place and insome cases the beers are produced by simply diluting full-strength beerswith water.

As the physical processes result in the evaporation, loss or dilution offermentation-derived flavor compounds (or a complete lack offermentation-derived flavor compounds as in the case with unfermentednon-alcoholic or alcohol-free beer), these types of beers are oftencharacterized as being less flavorful than full-strength beer or mayhave an undesirable wort-like taste (Zufall and Wackerbauer. 2000).

German patent DD 288619 A5 describes the use of Pichia farinosa yeaststrains to ferment unhopped beer wort to obtain an alcohol-free beveragewith fruity aroma.

However, there exists a need for improved methods for preparing alow-alcohol or alcohol-free fermented beverage wherein the presence ofdesirable flavor compounds, such as esters and higher alcohols, isenhanced.

SUMMARY OF THE INVENTION

The present invention is based on the surprising findings by theinventors that Pichia kluyveri yeast strains, only capable of fermentingthe glucose in beer wort, is capable of producing high concentrations ofdesirable flavor compounds with only little ethanol production.

The brewing yeasts of Saccharomyces pastorianus, Saccharomycescarisbergensis and Saccharomyces cerevisiae utilize both glucose as wellas maltose, the main sugars in wort (Boulton and Quain. 2010) resultingin the formation of alcohol and flavor compounds during fermentation.The higher the concentration of wort sugars, the more alcohol and flavorcompounds will be produced. In order to produce less alcohol, a solutionis to use a yeast that only ferments glucose and not maltose, therebyonly producing alcohol from the glucose. Glucose is present in muchlower concentrations than maltose in wort (Table 1) and therefore lessalcohol will be produced from the same amount of wort compared to when aconventional brewing yeast, e.g. Saccharomyces pastorianus orSaccharomyces carisbergensis, is used. These types of yeasts exist andexamples include certain Saccharomyces and non-Saccharomyces yeaststrains, such as Saccharomycodes ssp (Liu et al. 2011).

However, only a relatively small amount of flavor will be produced bythese yeast strains from the limited amount of glucose resulting in abeer with lower concentrations of key flavor compounds than beers withmore than 4% (vol/vol) alcohol.

TABLE 1 Typical sugar spectrum of wort (Stewart. 2010) Percentcomposition Glucose 10-15 Fructose 1-2 Sucrose 1-2 Maltose 50-60Maltotriose 15-20 Dextrins 20-30

One way of solving this problem, is to identify a highly aromatic yeast,which is not able to utilize the maltose, in order to produce highconcentrations of relevant fermentation-derived flavor compounds fromlimited amounts of glucose, resulting in similar or higherconcentrations of key flavor compounds than when the full fermentationtakes place. The inventors of the present invention have found that byusing this method and Pichia kluyveri yeast strains, it is possible toproduce alcohol-free beer and low-alcohol beer (according to EUlegislation) with the same flavor as standard, full-strength beers. Thisis not possible using conventional brewing yeast such as Saccharomycescerevisiae and Saccharomyces pastorianus or Saccharomycescarlsbergensis, dilution, physical alcohol removal processes or beersproduced form non-fermented substrates.

Accordingly, a first aspect of the invention relates to a method ofpreparing a low-alcohol or alcohol-free beverage comprising the stepsof:

-   -   a) providing a wort; and    -   b) fermenting the wort with at least one Pichia kluyveri yeast        strain to obtain a low-alcohol or alcohol-free beverage.

An additional aspect of the present invention relates to a low-alcoholor alcohol-free beverage obtainable by the method according to the firstaspect of the invention.

A further aspect of the present invention relates to a low-alcohol oralcohol-free beverage, wherein the low-alcohol or alcohol-free beveragehas an isoamyl acetate content of at least 0.5 ppm (mg/L).

An even further aspect of the present invention relates to use of aPichia kluyveri yeast strain for preparing low-alcohol or alcohol-freebeverage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an optimized beer brewing process for Pichia kluyveribeer. Comparison of the standard (left) versus Pichia kluyveri (right)brewing process. Steps that are different from the standard brewingprocess are highlighted.

FIG. 2 illustrates the flavor profile comparison of 5 differentalcohol-free beers: 3 horses. Hollandia, Bavaria (all with 0% alcohol)and the beers produced with Pichia kluyveri strain A (0.1% (vol/vol)alcohol) and strain B (0.2% (vol/vol) alcohol). Carlsberg pilsner beerwith 4.6% (vol/vol) alcohol was used as a reference pilsner beer and twoother pilsner beers are shown as well: Stella (5.2% (vol/vol) alcohol)and Heineken (5.0% (vol/vol) alcohol). 5 different flavor compounds weremeasured with headspace-GC-FID: isoamyl acetate, isoamyl alcohol, ethylbutyrate, ethyl hexanoate and ethyl octanoate. The flavor compoundconcentrations in Carlsberg pilsner beer were used as a reference andwere scaled to be 1. The flavor concentrations in all beers were put onthe same scale in order to be able to compare the values.

FIG. 3 shows flavor profile comparison of 4 different beers:alcohol-free beer with Pichia kluyveri strain A (0.1% (vol/vol)alcohol), low alcohol beer with Pichia kluyveri strain A (0.7% (vol/vol)alcohol), Carlsberg pilsner beer (4.6% (vol/vol) alcohol) which was usedas a reference pilsner beer (control) and malt beer (1.8% (vol/vol)alcohol). 5 different flavor compounds were measured withheadspace-GC-FID: isoamyl acetate, isoamyl alcohol, ethyl butyrate,ethyl hexanoate and ethyl octanoate. The flavor compound concentrationsin Carlsberg pilsner beer were used as a reference and were scaled tobe 1. The flavor concentrations in the 3 beers were put on the samescale in order to be able to compare the values.

FIG. 4 illustrates the diacetyl concentrations in beer trials withPichia kluyveri strain A and a Saccharomyces cerevisiae beer yeast.

FIG. 5 depicts yeast cell growth during fermentation.

FIG. 6 shows isoamyl acetate ethyl decanoate and phenylethyl acetateconcentrations in all fermentation products with Saccharomycodesludwigii and Pichia kluyveri, as well as in the basic wort (with andwithout hops). w/o=without.

FIG. 7 illustrates isobutyl acetate and butyl acetate concentrations inall fermentation products with Saccharomycodes ludwigii and Pichiakluyveri, as well as in the basic wort (with and without hops).w/o=without.

FIG. 8 shows isobutanol, 1-butanol and isoamyl alcohol concentrations inall fermentation products with Saccharomycodes ludwigii and Pichiakluyveri, as well as in the basic wort (with and without hops).w/o=without.

FIG. 9 depicts hexanoic, octanoic and decanoic acid concentrations inall fermentation products with Saccharomycodes ludwigii and Pichiakluyveri, as well as in the basic wort (with and without hops).w/o=without.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have unexpectedly found thatPichia kluyveri yeast strains are able to enhance the presence ofdesirable flavor compounds in beverages prepared by fermenting wort withthe Pichia kluyveri yeast strains. In particular, it was found thatconcentrations of flavor compounds desirable for beer, such as isoamylacetate, isoamyl alcohol and ethyl octanoate, were increased.

Surprisingly, this enhancement of beer flavor compounds is present whenyeast strains of the species Pichia kluyveri are used in the absence ofconventional brewing yeast strains, such as yeast strains of the generaSaccharomyces and Brettanomyces.

The process of brewing beer is well known to the skilled person and canbe outlined in the following way (see FIG. 1); malt is prepared fromdried, germinated cereal grains (mainly barley or wheat) and groundedinto a grist which may contain unmalted adjuncts. The grist is mashed(mixed with water and steeped) to allow enzymes in the malt to convertthe starch into sugars. The grain particles and adjuncts are separatedfrom the liquid wort in a process called lautering. The malt making andmashing steps can be skipped by adding water to malt extract. Afteraddition of hops and/or other ingredients such as herbs and sugars, thewort is boiled (hops may also be added after boiling), cooled andaerated. The wort is then moved to a fermentation tank and fermented bythe addition of a brewer's yeast. The primary fermentation, lastingtypically 5 to 10 days, may be followed by a secondary fermentation stepusing a brewer's yeast. After fermentation the fresh beer or “green”beer, is conditioned, optionally filtrated and carbonated. The beer isfilled on bottles and optionally pasteurized.

Hops can be added to the wort to balance the sweetness of the malt withbitterness and impart onto the beer desirable flavors and aromas.Several varieties exist including but not limited to Ahtanum, Amarillo,Apollo, Bravo, Calypso, Cascade, Centennial, Chelan, Chinook, Citra,Cluster, Columbus, Comet, Crystal, El Dorado, Eroica, Galena, Glacier,Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier,Mosaic, Newport, Nugget, Palisade, San Juan, Santiam, Satus, Simcoe,Sonnet Golding, Sterling, Summit, Super Galena, Tillicum, Tomahawk,Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold,Bramling Cross, Bullion, Challenger, First Gold, Fuggles, Goldings,Herald, Northdown, Northern Brewer, Phoenix, Pilgrim, Pilot, Pioneer,Progress, Target, Whitbread Golding Variety (WGV), Hallertau,Hersbrucker, Saaz, Tettnang, Spalt, Ella, Feux-Coeur Francais, Galaxy,Green Bullet, Hallertau Aroma, Kohatu, Motueka, Nelson Sauvin, PacificGem, Pacific Jade, Pacifica, Pride of Ringwood, Rakau, Riwaka, SouthernCross, Sticklebract, Summer, Super Alpha, Super Pride, Topaz, Wai-iti,Hallertau Herkules, Hallertau Magnum, Hallertau Taurus, Magnum, Merkur,Opal, Perle, Saphir, Select, Smaragd, Tradition, Bor, Junga, Lublin,Marynka, Premiant, Sladek, Strisselspalt, Styrian Atlas, Styrian Aurora,Styrian Bobek, Styrian Celeia, Styrian Golding, Sybilla, Tardif deBourgogne and Sorachi Ace.

The method for preparing a low-alcohol or alcohol-free beverage of thepresent invention comprises the steps of:

-   -   a) providing a wort, and    -   b) fermenting the wort with at least one Pichia kluyveri yeast        strain to obtain a low-alcohol or alcohol-free beverage.

The term “low-alcohol beverage” herein is defined as a liquid fordrinking with an alcohol content of more than 0.5% (vol/vol) of alcoholand no more than 1.2% (vol/vol) of alcohol.

The term “alcohol-free beverage” herein is defined as a liquid fordrinking with an alcohol content of no more than 0.5% (vol/vol) ofalcohol.

The term “wort” herein has the conventional meaning in the art andrefers to the sugary liquid extracted from the mashing process of beerbrewing.

The concentration of glucose in the wort is critical for the resultingalcohol percentage of the beverage and can be adjusted by changing themashing process, by addition of enzymes and by other methods known tothe skilled person.

In a preferred embodiment the wort has a concentration of glucosebetween 0.01% to 3% (w/vol), such as between 0.5% to 1.0% (w/vol).

Preferably, the fermentation in step b) is carried out under sterileconditions (controlled fermentation) and with Pichia kluyveri as theonly species of yeast strain added.

The skilled person will readily be able to determine a concentration ofinoculation of the Pichia kluyveri yeast strain suitable for preparationof a low-alcohol or alcohol-free beverage according to the method.

In a preferred embodiment, the Pichia kluyveri yeast strain isinoculated in a concentration of at least 1×10⁴ CFU/ml (colony-formingunits/ml), such as at least 5×10⁴ CFU/ml, such as at least 1×10⁵ CFU/ml,such as at least 5×10⁵ CFU/ml, such as at least 1×10⁶ CFU/ml, such 10 asat least 5×10⁶ CFU/ml.

In another preferred embodiment the method further comprises the stepa1) adding to the wort at least one hop variety prior to fermenting thewort with the at least one Pichia kluyveri yeast strain.

In another preferred embodiment the fermentation of the wort with the atleast one Pichia kluyveri yeast strain is carried out for at least 7days, such as for at least 14 days, such as for at least 21 days, suchas for at least 28 days.

In another preferred embodiment the fermentation of the wort with the atleast one Pichia kluyveri yeast strain is carried out at a temperatureof between 18° C. and 22° C.

In yet another preferred embodiment the method further comprises thestep c) bottling and bottle pasteurizing the low-alcohol or alcohol-freebeverage.

Preferably, the at least one Pichia kluyveri yeast strain is capable offermenting glucose but not maltose or maltotriose. This can be tested bygrowing the yeast strain on a culture medium with only maltose ormaltotriose.

In a preferred embodiment the at least one Pichia kluyveri yeast strainis selected from the group consisting of the Pichia kluyveri PK-KR1(JT1.28; strain A), that was deposited on 24 Aug. 2006 at the NationalMeasurement Institute, 541-65 Clarke Street, South Melbourne, Victoria3205, Australia, by University of Auckland, School of BiologicalSciences, Auckland 1142, New Zealand, under accession numbersV06/022711, and the Pichia kluyveri PK-KR2 (JT3.71), that was depositedon 24 Aug. 2006 at the National Measurement Institute, 541-65 ClarkeStreet, South Melbourne, Victoria 3205, Australia, by University ofAuckland, School of Biological Sciences, Auckland 1142, New Zealand,under accession numbers V06/022712.

Preferably the low-alcohol or alcohol-free beverage is a low-alcohol oralcohol-free beer.

The term “beer” as used herein refers at least to beers prepared frommashes prepared from malted cereals as well as mashes prepared fromunmalted cereals, and mashes prepared from a mixture of malted andunmalted cereals. The term “beer” also refers to beers prepared withadjuncts and beers with all possible alcohol contents.

The term “low-alcohol beer” herein refers to a beer with an alcoholcontent of more than 0.5% and no more than 1.2% (vol/vol) of alcohol.

The term “non-alcohol beer” herein refers to a beer with an alcoholcontent of no more than 0.5% ABV.

An additional aspect of the present invention relates to a low-alcoholor alcohol-free beverage obtainable by the method described herein.

In a preferred embodiment the low-alcohol or alcohol-free beverage is alow-alcohol or alcohol-free beer.

A further aspect of the present invention is directed to a low-alcoholor alcohol-free beverage, wherein the beverage has an isoamyl acetateconcentration of at least 0.5 ppm (parts per million; mg/L), such as atleast 0.6 ppm, such as at least 0.7 ppm, such as at least 0.8 ppm, suchas at least 0.9 ppm, such as at least 1.0 ppm, such as at least 1.2 ppm,such as at least 1.4 ppm, such as at least 1.6 ppm, such as at least 1.8ppm, such as at least 2.0 ppm, such as at least 2.5 ppm, such as atleast 3.0 ppm, such as at least 3.5 ppm, such as at least 4.0 ppm, suchas at least 4.5 ppm, such as at least 5.0 ppm.

Preferably, the low-alcohol or alcohol-free beverage has an ethyloctanoate concentration of at least 10 ppb (parts per billion; pg/L),such as at least 11 ppb, such as at least 12 ppb, such as at least 13ppb, such as at least 14 ppb, such as at least 15 ppb, such as at least20 ppb, such as at least 25 ppb, such as at least 30 ppb, such as atleast 40 ppb, such as at least 50 ppb, such as at least 60 ppb, such asat least 70 ppb, such as at least 80 ppb, such as at least 90 ppb, suchas at least 100 ppb.

In a preferred embodiment the beverage has an alcohol concentration ofmore than 0.5% (vol/vol) alcohol and no more than 1.2% (vol/vol)alcohol.

In another preferred embodiment the beverage has an alcoholconcentration of no more than 0.5% (vol/vol) alcohol.

Preferably, the low-alcohol or alcohol-free beverage is a low-alcohol oralcohol-free beer.

The present invention in one further aspect relates to use of a Pichiakluyveri yeast strain for preparing a low-alcohol or alcohol-freebeverage.

In a preferred embodiment the low-alcohol or alcohol-free beverage is alow-alcohol or alcohol-free beer.

EXAMPLES Example 1

Materials and Methods

Fermentation Set-Up

Both low alcohol and alcohol-free beer were produced with 2 differentPichia kluyveri strains (strain A and strain B). In total 2 trials havebeen carried out:

-   -   1) Alcohol-free beer with Pichia kluyveri strain A and strain B        compared to each other.    -   2) Low alcohol beer with Pichia kluyveri, strain A.

The brewing recipe was initially the same for both trials, except forthe hop addition. The alcohol-free beer was produced on a 1500 literscale. The low alcohol beers with Pichia kluyveri, strain A and strainB, were produced on a 1000 liter scale. The basis for all the beers wasa mixture of 4 different kinds of malt: pilsner malt 20%, wheat malt38%, Munich malt 38% and Cara 50 malt 4%. The initial sugarconcentration was measured to 8.3° Plato with an Anton Paar BeerAlcolyzer.

All yeast strains were inoculated at 5×10⁶ CFU/ml.

For the alcohol-free beer, with a comparison between strain A and strainB of Pichia kluyveri, only hop extract was used for the bitterness. Thefermentation was carried out at 20° C. for about three weeks.

For the low alcohol beer, hop extract was used for the bitterness and atthe end of wort cooking, Tettnang and Amarillo hops were used as aromahops. The fermentation was carried out at 21° C. The fermentation wascarried out for three weeks, and in the middle of fermentation, Tettnangand Amarillo hops were added as hop bells for dry hopping. Thefermentation temperature was lowered to 4° C. in the course of the dryhopping.

After fermentation, all beers were cooled down to 4° C. for severaldays, filtered and pasteurized. The beer was then filled in bottles.

The initial glucose concentration was >1% in the first trial, but around0.5% in the last two trials and the glucose concentration decreased veryslowly during fermentation. At the end of fermentation, almost allglucose was consumed by the yeast and converted to ethanol.

An overview of the brewing process is given in FIG. 1, where it iscompared to a normal brewing process. Important steps in the ‘new’brewing process are:

-   -   1) Regulating the mashing conditions to optimize the glucose        concentration in the wort    -   2) Assure that the hop added is sterile, so using hop pellets or        hop extracts, no dry hopping, as this can result in        contamination of the final beer    -   3) A longer fermentation time to optimize the flavor profile of        the beer    -   4) Bottle pasteurization to avoid the risk of contamination due        to the high maltose level in the final beer.

As Pichia kluyveri strains are very slow fermenting strains, it isimportant to keep the wort and the beer sterile. Any addition of hopsshould be made in a sterile manner, meaning at least 30 min. before theend of boiling of the wort. Dry hopping (addition of hops after boiling)should not be used for the production of low-alcohol or alcohol-freebeers, as there is a risk of contaminating the beer. As a lot of sugaris left in the final beer, extra care should be taken to avoid spoilage.

Lab Brewing Trial

Lab-scale fermentation trials were carried out in 500 ml of wort. Thewort was prepared with wheat malt extract (Brewferm). The malt extractwas mixed with water to reach an initial sugar content of 10° P (Plato)as measured with an Anton Paar Beer Alcolyzer. Amarillo hop pellets wereadded in a closed coffee filter to the wort and this was boiled for 30min. to extract the hop flavor and to add bitterness to the beer. Hoppellets were added to reach 23 EBU (European Bitterness Units). Afterboiling the, the coffee filter was removed from the wort and the wortwas transferred to 1 L bottles, which were closed by water locks.Fermentation was carried out at 20° C. After 5 days of fermentation asample was taken for diacetyl measurement.

Flavor Analysis

Headspace gas chromatography coupled with flame ionization detection(HSGC-FID) is used for the measurement of acetate esters, ethyl estersand higher alcohols in the samples. Samples are analyzed with acalibrated Perkin Elmer GC System with a headspace sampler. The GC isequipped with a HP-FFAP column (25 m×0.20 mm×0.33 μm, AgilentTechnologies, Germany). The split-splitless injector is used and held at180° C. Samples are heated for 30 min. at 70° C. in the headspaceautosampler before injection (needle temperature: 110° C.). Helium isused as the carrier gas. The SOF-program works as follows: afterstarting at 60° C., the oven temperature is raised after 2 min. from 60°C. to 230° C. at 45° C./min and is finally held at 230° C. for 5 min.During the GC-program a constant flow rate (10 mL/min) of the carriergas (He) is maintained. The FID temperature is kept constant at 220° C.The results are analyzed with Turbochrom software. Headspace GC-MS wasused for the analysis of diacetyl. In principal, this method is the sameas the method used to detect flavor compounds, but an extra splittransports additional helium, leading to an increased flow rate andresulting in a higher concentration of the compound on the column.Further differences were that the pressurized vial acts for 0.03 min. asa carrier gas reservoir and the GC program starts at a temperature of90° C.

Ethanol and Density Analysis

Ethanol and density analysis was performed with an alcolyzer beeranalyzing system from Anton-Paar.

Results

TABLE 2 Concentrations (in ppm; mg/L) of ethyl butyrate, isoamylacetate, isoamyl alcohol, ethyl hexanoate and ethyl octanoate in 6commercial beers and beer prepared with Pichia kluyveri strain A andPichia kluyveri strain B. Ethyl Isoamyl Isoamyl Ethyl Ethyl butyrateacetate alcohol hexanoate octanoate Carlsberg 1.10 1.00 34.0 0.04 0.10Stella 2.14 0.71 50.0 0.11 0.10 Heineken 2.02 4.87 46.0 0.10 0.09 3horses 0.00 0.00 0.0 0.00 0.00 Hollandia 0.00 0.26 3.2 0.00 0.00 Bavaria0.00 0.18 2.0 0.00 0.00 Malt beer 0.71 0.21 11 0.01 0.01 Pichia A - 0.431.96 2.00 0.03 0.12 alcohol-free Pichia B - 0.26 4.94 2.00 0.07 0.13alcohol-free Pichia A - 0.27 2.50 1.80 0.02 0.13 low alcohol

Alcohol-Free Beer Produced by the Use of Pichia kluyveri DuringFermentation

To produce an alcohol-free beer, a brewing process with Pichia kluyveriyeast strains was carried out (see Materials and methods). The Pichiakluyveri strains were used to produce a beer with an alcohol percentageless than 0.5%. In European terms, this is considered a non-alcoholicbeer. This brewing trial was performed in a 1000 liter scale, withPichia kluyveri strain A and strain B and a slightly lower fermentationtemperature of 20° C. (see FIG. 1 for brewing process details). All theglucose was consumed at the end of fermentation and the beer producedwith strain A had an alcohol percentage of 0.1%. The beer produced withstrain B had an alcohol percentage of 0.2%. The fermentation time wasthree weeks.

The two final beers were used for flavor analysis. In order to have anidea of the flavor profile of commercial alcohol-free beers, 4commercial alcohol-free beers were included in the flavor analysis. The3 beers were 3 horses, Hollandia and Bavaria. The following ingredientswere mentioned on the beers:

-   -   3 horses classic non-alcoholic beer: water, barley, malt, maize        and hops.    -   Hollandia malt non-alcoholic beer: water, barley, malt, maltose        and hop extract.    -   Bavaria non-alcoholic beer: pure mineral water, barley malt,        maltose and hops.

As control, the flavor profile of a Carlsberg pilsner beer (4.6%(vol/vol) alcohol) was used. To compare the trial beers brewed withPichia kluyveri with other commercial pilsner beers, the flavor profileof 2 other pilsner beers was measured: Stella premium lager beer (5.2%(vol/vol) alcohol) and Heineken lager beer (5.0% (vol/vol) alcohol).

As can be seen from the list above, the main ingredients for thealcohol-free beers are water, malt (sometimes with unmalted barley asadjunct) and hops. These are the same main ingredients as in the trialswith Pichia kluyveri.

The result of the flavor analysis is depicted in FIG. 2. Theconcentrations of the 5 flavor compounds isoamyl acetate, isoamylalcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate are shown,each scaled so that the concentration of the respective flavor compoundfor the Carlsberg pilsner beer is 1.

FIG. 2 clearly shows that all the commercial alcohol-free beers have avery low concentration of typical beer flavor compounds, when comparedto the Carlsberg pilsner beer and compare to the other three pilsnerbeers. However, the beer produced with Pichia kluyveri has similarlevels of specific beer flavor compounds as the reference pilsner beer,in particular isoamyl alcohol, ethyl hexanoate and ethyl octanoate.Ethyl butyrate was present in lower amount (half the concentration) andisoamyl acetate was present in higher amounts (double or moreconcentration). When compared to the other pilsner beers, it seems thatthe isoamyl acetate concentration in the Pichia kluyveri beer is stillhigher, as well as ethyl octanoate concentrations. However, ethylbutyrate and ethyl hexanoate concentrations were higher in the pilsnerbeers than in the Pichia kluyveri beer. These compounds are present invery low concentrations in beer and are less important compared toisoamyl acetate concentrations. Isoamyl alcohol concentrations does notdiffer much between the Pichia kluyveri beer and the pilsner beers, butwas virtually absent in the alcohol-free beers. When the overall flavorprofile of the Pichia kluyveri beer is compared to to the alcohol-freeand pilsner beers, it seems that the flavor profile of the Pichiakluyveri beer is closer to a beer with 4.6-5.2% (vol/vol) alcohol thanthe flavor profile of any of the commercial alcohol-free beers measured.

As these flavor compounds have a similar fruity flavor, they work insynergy. Tasting of the Pichia kluyveri beer revealed that the flavorwas very beer-like and this beer was preferred over the commercialalcohol-free beers by a tasting panel consisting of brewers and beerconsumers.

Low-Alcohol Beer Produced with Pichia kluyveri

This beer was brewed from 1500 liter wort and the fermentationtemperature was 21° C. (see FIG. 1 for brewing process details). Thistrial was carried out with Pichia kluyveri strain A. All the glucose wasconsumed at the end of fermentation and the final ethanol level was0.7%. The fermentation time was three weeks.

As can be seen from FIG. 3, both the alcohol-free and low-alcohol beerprepared with Pichia kluyveri strain A have a similar flavor profile,meaning that both the alcohol-free and low-alcohol beer are very closeto a normal pilsner beer flavor profile. Again the main esters isoamylacetate, ethyl butyrate, ethyl hexanoate, ethyl octanoate and isamylalcohol determine the flavor profile.

Measurement of Diacetyl Produced by Pichia kluyveri

To be sure that Pichia kluyveri does not produce any significantoff-flavors in beer, the production of the most important off-flavor inbeer, diacetyl, was measured in a lab brewing trial with Pichia kluyveristrain A only, compared to a beer with a Saccharomyces cerevisiae beerstrain (FIG. 4).

It is clear from FIG. 4 that Pichia kluyveri produces much less diacetylcompared to a Saccharomyces cerevisiae brewing strain. This is importantas diacetyl is often regarded as an off-flavor in beer. Especially, inalcohol-free and low-alcohol beers where no alcohol is present, thetaste threshold level of diacetyl is probably much lower than in normalstrength beers.

Conclusion

The flavor profile results of the alcohol-free and low-alcohol beersproduced with Pichia kluyveri show that this yeast strain is ideallysuited for the production of beers with lower or no alcohol. Especially,the production of a high amount of esters, coming from a very lowglucose concentration is a special property seen in Pichia kluyveristrains. Together with isoamyl acetate, other esters are enhanced, whichgives the beers with low or no alcohol a beer-like flavor profile.

Example 2

Saccharomycodes ludwigii has been mentioned in Branyik et al. (2012) asthe most successful genus, other than Saccharomyces, used for theindustrial production of alcohol-free beer.

To compare the properties of Pichia kluyveri with the properties ofSaccharomycodes ludwigii for preparation of low-alcohol beer a secondexperiment was set up:

Materials and Methods

Culturing Yeast

The two yeast cultures (Pichia kluyveri strain A and a Saccharomycodesludwigii yeast strain) were prepared in YPD (2% glucose, 1% peptone and0.5% yeast extract), with an adjusted pH of 5.5. The YPD medium wasautoclaved at 121° C. for 20 minutes.

The yeast cultures were plated out on YGC plates (Sigma-Aldrich) and asingle colony was inoculated in 5 ml YPD medium. These cultures weregrown overnight in a shaking incubator at 30° C. and inoculated in 500ml YPD. After 2 days, a sample was taken for cell counts to be able toinoculate the yeast cultures at the same initial cell count in the wortmedium.

Lab Brewing Trial

Lab-scale fermentation trials were carried out in 500 ml of wort. Thewort was prepared with pilsner malt extract (Brewferm). The malt extractwas mixed with water to reach an initial sugar content of 7° P (Plato)as measured with an Anton Paar Beer Alcolyzer. In total, 10 liter wortwas prepared in this way.

In the first set-up, the wort was boiled for 60 minutes without additionof hops. In the second set-up,

4.5 g of Amarillo, 4.5 g of Cascade and 2 g of Tettnanger hop pelletswere added to the wort and boiled for 60 minutes to give bitterness. 10minutes before the end of boiling, 1.5 g Cascade, 1.5 g Amarillo and 1 gTettnanger hop pellets were added as aroma hops. After boiling, the wortwas transferred to 2 L bottles (1600 ml of wort per bottle), which wereclosed by water locks.

The yeast cultures were inoculated at 1×10⁶ CFU/ml and fermentation wascarried out at 20° C. After 5 days of fermentation, a sample was takenfor flavor analysis.

Ethanol Analysis

Ethanol was measured with the Ethanol Enzymatic Bioanalysis kit ofBoehringer Mannheim.

Flavor Analysis

Flavor analysis was carried out at Laboratorio de Analisis del Aroma yEnologia (Zaragoza, Spain). Minor aroma compounds are expressed in μg/L(ppb) and are measured with solid-phase extraction and Gaschromatography with mass spectrometric detection, while major aromacompounds are expressed in mg/L (ppm) and are measured with gaschromatographic-flame ionization detection after extracting thecompounds in dichloromethane.

Results

Lab-scale fermentations were carried out in two types of pilsner wort:wort without the addition of hops and the same pilsner wort withaddition of bitter and aroma hops (Cascade, Amarillo and Tettnanger)(see materials and methods). Two different yeast strains were tested:Saccharomycodes ludwigii and Pichia kluyveri. The yeast cultures wereinoculated at a cell count of 1E6 CFU/ml after culturing them in YPDmedium. In total, 4 different fermentations were carried out:

1) Saccharomycodes ludwigii in wort without hops (S. ludwigii w/o hops)

2) Saccharomycodes ludwigii in wort with hops (S. ludwigii with hops)

3) Pichia kluyveri in wort without hops (P. kluyveri w/o hops)

4) Pichia kluyveri in wort with hops (P. kluyveri with hops)

All fermentations were carried out for 5 days at 20° C.

During the fermentation, cell counts were measured every day to followyeast growth (FIG. 5).

It is clear from FIG. 5 that both yeast strains survived in the wortwith and without hops. Pichia kluyveri is clearly growing, compared toSaccharomycodes ludwigii, which has a stable cell count during the wholefermentation.

After 5 days, ethanol concentration was measured in all fermentationproducts (Table 3).

TABLE 3 Ethanol concentration in all 4 fermentation productsFermentation product Ethanol %/v/v) S. ludwigii w/o hops 0.3 C. ludwigiiwith hops 0.3 P. kluyveri w/o hops 0.1 P. kluyveri with hops 0.1

Table 3 shows that both Saccharomycodes ludwigii and Pichia kluyveriproduce very low amounts of ethanol. Pichia kluyveri seems better atproducing no alcohol, as this strain grows to higher cell counts, butproduces less alcohol, compared to Saccharomycodes ludwigii.

The fermentation products at day 5 were also analyzed for flavor profile(FIGS. 6, 7, 8 and 9). The most important flavor compounds for beer areesters and higher alcohols.

FIGS. 6 and 7 show the ester concentrations in all final fermentationproducts, as well as in the basic wort with and without hops. All esterconcentrations are the highest in the fermentation products with Pichiakluyveri, both with and without hops. In the case of isoamyl,phenylethyl, isobutyl and butyl acetate, Pichia kluyveri produces higheramounts of these esters when no hops are added, compared to ethyldecanoate. Especially the levels of isoamyl acetate and phenylethylacetate are very high. Isoamyl acetate results in a banana aroma, whilephenylethyl acetate results in a more flowery, honey aroma. Both estercompounds are wanted compounds in beer. It has to be noted that theresults depicted here are ester concentrations after 5 days offermentation, and these concentrations will decrease over time in afinal beer. It is important to have high ester concentrations to startwith, resulting in a fruity beer for a longer period of time, as theseester compounds are hydrolyzed over time in the final beer.

The fusel alcohol concentrations, depicted in FIG. 8, are quite similarbetween the fermentation products of Saccharomycodes ludwigii and Pichiakluyveri. Only isoamyl alcohol concentrations are higher in thefermentation products with Saccharomycodes ludwigii, probably becausethis compound is not converted to isoamyl acetate, as is the case in thePichia kluyveri fermentation products.

In FIG. 9, three acids are depicted, which are negative flavor compoundsin beer. Especially decanoic acid gives a rancid, cheesy flavor to beerand is therefore unwanted. It is very clear that especiallySaccharomycodes ludwigii is producing high amounts of these acids,making it less suitable for beer production.

Conclusion

The lab brewing trial with the two different non-Saccharomyces yeaststrains clearly indicates that Pichia kluyveri is better suited for theproduction of alcohol-free beer, compared to Saccharomycodes ludwigii.Pichia kluyveri produces less alcohol, more wanted ester compounds andless unwanted acids, compared to Saccharomycodes ludwigii.

Deposits

The Pichia kluyveri PK-KR1 (JT1.28 or strain A) and PK-KR2 (JT3.71)strains were deposited on 24 Aug. 2006 at the National MeasurementInstitute, 51-65 Clarke Street, South Melbourne, Victoria 3205,Australia, by University of Auckland, School of Biological Sciences,Auckland 1142, New Zealand, and given the accession numbers V06/022711and V06/022712 as described in WO 2009/110807.

REFERENCES

DD 288619

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Branyik, T, Silva, D. P., Baszczyn, M., Lehnert, R., Almeida e Silva, J.B., 2012. A review of methods of low alcohol and alcohol-free beerproduction. Journal of Food Engineering 108,493-506.

Liu, Y., Li, H., Du, J.-H. 2011. Non-alcoholic Beer Production bySaccharomycodes ludwigii. Food Science, 32: 186-190.

Montanari, L., Marconi, O., Mayer, H., Fantozzi, P., 2009. Production ofalcohol-free beer. In: Preedy, V. R. (Ed.), Beer in Health and DiseasePrevention. Elsevier Inc., Burlington, Mass., 61-75.

Saerens S. M., Delvaux F. R., Verstrepen K. J., Thevelein J. M. 2010.Production and biological function of volatile esters in Saccharomycescerevisiae. Microbial Biotechnology 3:165-77.

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Swiegers, J. H., S. M. G. Saerens, I. S. Pretorius. 2007. Thedevelopment of yeast strains as tools to adjust the flavor of fermentedbeverage to market specifications. Havkin D. and Belanger F.Biotechnology in flavor production. Blackwell Publishing Ltd.

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1-9. (canceled)
 10. A low-alcohol or alcohol-free beer with an alcoholcontent of no more than 1.2% (vol/vol) having an isoamyl acetate contentof at least 0.5 ppm (mg/L) which comprises Pichia kluyveri as the onlyspecies of yeast strain added.
 11. A low-alcohol or alcohol-free beeraccording to claim 1, having an alcohol content of no more than 0.5%.12. A low-alcohol or alcohol-free beer according to claim 1, wherein thePichia kluyveri strain is selected from Pichia kluyveri strain PK-KR1having accession number V06/022711 and Pichia kluyveri strain PK-KR2having accession number V06/022712.
 13. A low-alcohol or alcohol-freebeer according to claim 1, wherein the beer has an ethyl octanoateconcentration of at least 10 μg/L.
 14. A low-alcohol or alcohol-freebeer according to claim 10, wherein the beer is hopped.
 15. A hoppedlow-alcohol or alcohol-free beer according to claim 14, wherein the hopis selected from Ahtanum, Amarillo, Apollo, Bravo, Calypso, Cascade,Centennial, Chelan, Chinook, Citra, Cluster, Columbus, Comet, Crystal,El Dorado, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty,Millenium, Mount Hood, Mount Rainier, Mosaic, Newport, Nugget, Palisade,San Juan, Santiam, Satus, Simcoe, Sonnet Golding,Sterling, Summit, SuperGalena, Tillicum, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus,Admiral, Brewer's Gold, Bramling Cross, Bullion, Challenger, First Gold,Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilgrim,Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV),Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Ella, Feux-CoeurFrancais, Galaxy, Green Bullet, Hallertau Aroma, Kohatu, Motueka, NelsonSauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Rakau,Riwaka, Southern Cross, Sticklebract, Summer, Super Alpha, Super Pride,Topaz, Wai-iti, Hallertau Herkules, Hallertau Magnum, Hallertau Taurus,Magnum, Merkur, Opal, Perle, Saphir, Select, Smaragd, Tradition, Bar,Junga, Lublin, Marynka, Premiant, Sladek, Strisselspalt, Styrian Atlas,Styrian Aurora, Styrian Bobek, Styrian Celeia, Styrian Golding, Sybilla,Tardif de Bourgogne, and Sorachi Ace.